Detection of parasite antigens in Leishmania infantum-infected spleen tissue by monoclonal antibody-, piezoelectric-based immunosensors.

Diseases such as leishmaniases are important causes of morbidity and mortality in Brazil, and their diagnoses need to be improved. The use of monoclonal antibodies has ensured high specificity to immunodiagnosis. The development of an immunosensor, coupling a monoclonal antibody to a bioelectronic device capable of quickly detecting Leishmania sp. antigens both qualitatively and quantitatively, is a promising alternative for the diagnosis of leishmaniasis due to its high specificity, low cost, and portability, compared with conventional methods. The present work was aimed at developing an immunosensor-based assay for detecting Leishmania infantum antigens in tissues of infected hosts. Four hybridomas producing monoclonal antibodies against L. infantum had their specificity confirmed by enzyme-linked immunosorbent assay. These antibodies were immobilized on a gold surface, covered with a thin film of 2-aminoethanethiol (cysteamine) and glutaraldehyde, blocked with glycine, and placed into contact with extracts of L. infantum -infected and noninfected control hamster spleens. The assay was able to detect 1.8 × 10(4) amastigotes/g of infected tissue. These results demonstrated that this assay may be useful for quantifying L. infantum amastigotes in organs of experimental animals for studies on pathogenesis and immunity and that it is a promising tool for the development of a diagnostic method, based on antigen detection, of human and dog visceral leishmaniasis.

A proteomic approach to identify proteins from Trichuris trichiura extract with immunomodulatory effects.

Infections with Trichuris trichiura and other trichurid nematodes have been reported to display protective effects against atopy, allergic and autoimmune diseases. The aims of the present study were to investigate the immunomodulatory properties of T. trichiura adult worm extract (TtE) and its fractions (TtEFs) on the production of cytokines by peripheral blood mononuclear cells and to identify their proteinaceous components. Fourteen TtEFs were obtained by ion exchange chromatography and tested for effects on cytokine production by peripheral blood mononuclear cells. The molecular constituents of the six most active fractions were evaluated using nano-LC/mass spectrometry. The homology between T. trichiura and the related nematode Trichinella spiralis was used to identify 12 proteins in TtEFs. Among those identified, fructose biphosphate aldolase, a homologue of macrophage migration inhibitory factor and heat-shock protein 70 may contribute to the immunomodulatory effects of TtEFs. The identification of such proteins could lead to the development of novel drugs for the therapy of allergic and other inflammatory diseases.

Inflammation and structural changes of splenic lymphoid tissue in visceral leishmaniasis: a study on naturally infected dogs.

The aim of this study was to identify splenic immuno-inflammatory patterns associated with natural infection by Leishmania chagasi. Spleen samples were obtained from 72 stray dogs from an endemic area of visceral leishmaniasis. The animals were grouped into four categories as follows: (i) potentially resistant to visceral leishmaniasis, with a positive leishmanin skin test result, and negative splenic culture for Leishmania parasites (ii) potentially susceptible to visceral leishmaniasis, with a negative leishmanin skin test and positive splenic culture for Leishmania (iii) infected with undefined susceptibility status, with a positive leishmanin skin test and positive splenic culture for Leishmania, and (iv) noninfected, with a negative leishmanin skin test, negative splenic culture for Leishmania, and negative serology for anti-Leishmania antibodies. Histopathological analyses showed that there was a higher frequency of perisplenitis (18/25, P < 0.0001), granuloma (7/25, P = 0.0102), structural disorganization (14/25, P < 0.0001), and atrophy of the lymphoid follicles (20/25, P = 0.0036) and of the marginal zone (15/25, P = 0.0025) in the potentially susceptible group than in the other groups. The data presented here show changes in the white pulp of the spleen that are associated with naturally acquired visceral leishmaniasis.

Associations among immunological, parasitological and clinical parameters in canine visceral leishmaniasis: Emaciation, spleen parasitism, specific antibodies and leishmanin skin test reaction.

Associations among parameters commonly used as markers of infection by Leishmania sp., or of susceptibility to visceral leishmaniasis, were investigated in 325 stray dogs from an area where this disease is endemic. Evidence of infection (presence of Leishmania in splenic cultures, positive leishmanin skin test (LST) or detection of anti-Leishmania antibody activity in the serum) was found in 57% of the animals. Both evidence of weight loss (chi(2)-test, P=0.0005) and presence of specific antibody activity in the serum (chi(2)-test, P<0.0001) were directly associated with positive splenic culture. The frequencies of animals with positive splenic culture were directly correlated with the intensities of antibody activity in the serum as measured by ELISA (relative risk of 3.4 for animals with moderate antibody levels and relative risk of 8.43 for animals with high-antibody levels). A negative association was observed between positive leishmanin skin test results and emaciation (chi(2), P=0.0089). Furthermore, animals with positive splenic cultures and negative leishmanin skin test results had higher levels of total serum IgG (Kruskal-Wallis test, P=0.001) and IgG2 (Kruskal-Wallis test, P=0.05) than animals with negative splenic cultures, and were more emaciated than animals with negative LST results and positive splenic cultures. The data presented herein suggest that associating these common parameters may improve their performance in predicting susceptibility to canine visceral leishmaniasis.

Dissociation between vasodilation and Leishmania infection-enhancing effects of sand fly saliva and maxadilan.

In this study, the ability of maxadilan and Lutzomyia longipalpis salivary gland lysate to enhance the infection of CBA mice by Leishmania major and of BALB/c mice by L. braziliensis was tested. No difference was observed between sizes of lesion in CBA mice infected with L. major and treated or not with salivary gland lysate or maxadilan, although they were injected in concentrations that induced cutaneous vasodilation. Although parasites were more frequently observed in foot pads and spleens of animals treated with maxadilan than in the animals treated with salivary gland lysate or saline, the differences were small and not statistically significant. The lesions in BALB/c mice infected with L. braziliensis and treated with maxadilan were slightly larger than in animals that received Leishmania alone. Such differences disappeared 14 weeks after infection, and were statistically significant only in one of two experiments.

Skin reactions to thimerosal and Leishmania in dogs from a leishmaniasis endemic area: it is better to keep them apart.

Positive Montenegro's skin test is a delayed type hypersensitivity reaction widely used as indicative of previous infection with Leishmania in both humans and dogs. Montenegro's antigen consists of a crude Leishmania antigen solution, usually containing thimerosal as preserving agent. In this work it is shown that a large proportion of dogs (11 out of 56) examined in an endemic area of leishmaniasis presented induration at the site of injection of a diluent containing thimerosal alone. This clearly demonstrates that thimerosal leads to a high number of false positive skin reactions in dogs and that its use in Montenegro's skin test antigenic preparations should be avoided.

A heart-specific CD4+ T-cell line obtained from a chronic chagasic mouse induces carditis in heart-immunized mice and rejection of normal heart transplants in the absence of Trypanosoma cruzi.

To study the role of autoreactive T cells in the pathogenesis of cardiomyopathy in Chagas' disease, we generated a cell line by repeated in vitro antigenic stimulation of purified splenic CD4+ T lymphocytes from a chronically Trypanosoma cruzi-infected mouse. Cells from this line were confirmed to be CD4+ CD8- and proliferated upon stimulation with soluble heart antigens from different animal species, as well as with T. cruzi antigen, in the presence of syngeneic feeder cells. In vitro antigen stimulation of the cell line produced a Th1 cytokine profile, with high levels of IFNgamma and IL-2 and absence of IL-4, IL-5 and IL-10. The cell line also terminated the beating of fetal heart clusters in vitro when cocultured with irradiated syngeneic normal spleen cells. In situ injection of the cell line into well established heart transplants also induced the cessation of heart beating. Finally, adoptive transfer of the cell line to heart-immunized or T. cruzi-infected BALB/c nude mice caused intense heart inflammation.

A strategy for the identification of T-cell epitopes on Leishmania cysteine proteinases.

In this study computational analysis was used to compile sequence alignments, construct a dendrogram and calculate physical data in order to predict potential T-cell epitopes of the Leishmania cysteine proteinase. Using multiple alignment of human and Leishmania proteinase sequences deposited on data bank sequences, it was possible to predict that the extreme C-terminus of cysteine proteinase (Cyspep, 355-444) contained three peptides (pI 361-370, pII 415-422 and pIII 431-444) with charge score, hydrophobicity and isoelectric points compatible for human leucocyte-associated antigen (HLA) class II binding. The prediction was confirmed in vitro through the ability of synthetic peptides corresponding to the predicted regions to stimulate peripheral blood mononuclear cells of patients with leishmaniasis.

Cohort study on canine emigration and Leishmania infection in an endemic area for American visceral leishmaniasis. Implications for the disease control.

American visceral leishmaniasis is a main public health matter in Brazil. Since dogs have been incriminated as the main urban reservoir of AVL agent Leishmania chagasi, a cohort study aimed at understanding the dynamics of the canine infection was carried out in Jequié--an endemic community in the Northeast of Brazil. The inhabited urban and periurban areas of Jequié were divided into 140 clusters of 0.25 km2. All 1681 dogs domiciled in 34 randomly selected clusters were screened for Leishmania antibodies in an enzyme-linked immunosorbent assay. After the seropositive dogs were painlessly eliminated, a cohort of 1286 seronegative dogs was followed up for 18 months, yielding a total of 1739.7 dog-years. The overall incidence of Leishmania infection, as assessed by the detection of Leishmania antibodies in blood samples collected every six months, was 6.55 cases/100 dog-years (95% confidence interval; CI 6.04-7.26). Two subsets of clusters, with 0.70 and 1.35 relative risks of infection, were identified. The annual emigration rate was 2.26 cases/100 dog-years (95% CI 1.86-2.66). The implications of these findings for the control of American visceral leishmaniasis are discussed.

Incomplete dominance of the low antibody response to Cryptosporidium parvum antigens in mice selected for high and low antibody responsiveness.

The humoral antibody response to Cryptosporidium was investigated in mice genetically selected for high (H) and low (L) antibody responsiveness. Groups of 4-5 mice from two different selections, general primary (GP) and general secondary (GS), were studied. Following immunization with Cryptosporidium parvum antigens, the maximum levels of IgG in the HGP (X +/- SD = 1.13 +/- 0.35, N = 5) in the HGS (0.42 +/- 0.15, N = 4) lines, and of IgM in the HGP line (0.86 +/- 0.53, N = 5) were significantly higher than those in their L counterparts (0.04 +/- 0.02, N = 5; 0.05 +/- 0.02, N = 4 and 0.24 +/- 0.07, N = 5, respectively). These findings were similar to those reported for other immunogens. However, the IgG (0.22 +/- 0.05, N = 4) and the IgM (0.33 +/- 0.08, N = 4) responses to immunization of F1 (LGP x HGP) hybrids indicated an incomplete dominance of the low response, in contrast to the incomplete dominance of the high response described for many other antigens and representing an important exception. In addition, the H, L and F1 mice did not develop detectable infections when inoculated with live Cryptosporidium oocysts, supporting the view that a reduced or zero antibody production itself is not enough to permit the establishment of Cryptosporidium infection in adult mice.

A cross-sectional serodiagnostic survey of canine leishmaniasis due to Leishmania chagasi.

Jequie, a community of about 144,500 inhabitants located in the State of Bahia, Brazil, is endemic for both visceral and cutaneous leishmaniases. In the present epidemiologic study, the urban and inhabited periurban areas of the town were divided into 140 clusters of 0.25 km2 each. The seroprevalence of canine Leishmania antibodies was investigated using an enzyme-linked immunosorbent assay as a screening test since its sensitivity was significantly higher than that of an indirect immunofluorescence assay. A total of 1,681 dogs was surveyed in 34 randomly sampled clusters. The overall prevalence of Leishmania antibodies in the dog population was 23.5%, with intracluster prevalences ranging from 0% to 67%. There was no correlation of these seroprevalences with the intracluster densities of canine populations, or with the distances from individual clusters to the town center. Moreover, the Leishmania transmission did not seem to follow any clear-cut spatial pattern, since large disparities in the seroprevalences of contiguous clusters were found. Curiously, human cases of visceral leishmaniasis have never been observed in some clusters with a relatively high prevalence of canine seroprevalences. Eight parasite isolates from seropositive dogs were found to belong to the same serodeme and zymodeme as Leishmania (L.) chagasi. The implications of these findings with respect to the epidemiology and control of American visceral leishmaniasis are discussed.

Circulating trans-sialidase activity and trans-sialidase-inhibiting antibodies in Trypanosoma cruzi-infected mice.

Parasite-derived trans-sialidase (TS) activity was demonstrated in the serum and blood of Trypanosoma cruzi-infected mice. Serum TS activity levels correlated well with parasitemia in BALB/c and Swiss mice during the initial stages of the infection. However, in later stages the TS activity levels decreased despite increasing parasitemia. This coincided with the appearance of circulating TS antibodies. On the other hand, there was always a good correlation between TS activity and parasitemia in athymic nude mice. Sera from mice with high parasitemia and low TS activity inhibited TS activity in vitro. The inhibition was also observed with purified serum IgG, and it was absorbed by staphylococcal protein A, indicating that it was caused by anti-TS IgG antibodies. These antibodies inhibited the enzymatic activity of insolubilized TS, indicating that they act by interfering with the catalytic site rather than by aggregating the enzyme. The presence of inhibitory antibodies, however, did not prevent the progression of parasitemia in BALB/c mice.

Role of sialic acid in the resistance of Trypanosoma cruzi trypomastigotes to complement.

Trypomastigotes of Trypanosoma cruzi, mammalian infective forms of the parasite, express an unusual cell surface trans-sialidase. This enzyme enables the parasite to rapidly sialylate its surface when supplied with alpha(2,3)-linked sialic acid from glycoconjugates in serum or on cell surfaces. Here we used a novel fluorescence-based, trypomastigote lysis assay to evaluate the role of sialic acid on the parasite's plasma membrane in providing protection against the complement cascade. Trypomastigotes were desialylated, and sialic acid removal was confirmed by a chemical assay and also by flow cytometry with the use of a mAb that recognizes a T. cruzi-sialylated epitope. Compared with sialylated trypomastigotes, which were completely refractory to lysis by human serum, only about 5% of the desialylated trypomastigotes were lysed by complement. However, further analysis revealed that the desialylated parasites had been resialylated during exposure to serum complement. Next we incubated desialylated trypomastigotes with samples of desialylated human serum. Although the sialidase-treated serum retained its full hemolytic activity, lysis of trypomastigotes increased only from 5 to 24%. This increase correlated with an enhanced deposition of complement protein C3 on the parasite surface. The ratio of C3b to lytically inactive iC3b was increased for desialylated, compared with sialylated, parasites. We conclude that although parasite sialic acid promotes C3b cleavage into iC3b, this mechanism alone does not account for the robust resistance of these parasites to complement lysis.

Combined occurrence of trypanosomal sialidase/trans-sialidase activities and leishmanial metalloproteinase gene homologues in Endotrypanum sp.

Endotrypanum (order Kinetoplastida: family Trypanosomatidae) is a parasite of forest dwelling tree sloths (Edentata: genera Choleopus and Bradypus). Unique among the haemoflagellates, this protozoan has an intraerythrocytic phase in the mammalian host. Nevertheless, many striking similarities exist between Endotrypanum and the human pathogen Leishmania that make it a useful model for epidemiological and evolutionary aspects of the biology of trypanosomatids. Importantly, Endotrypanum species share both the insect vector and host reservoir with certain species of Leishmania (subgenus Viannia). Because mixed infections with Endotrypanum and Leishmania are common in sloths and, therefore, likely to occur in the sandfly vector, there is a need for adequate biochemical markers to distinguish Endotrypanum from Leishmania infections. In this paper we show that Endotrypanum promastigotes possess sialidase and trans-sialidase activities, which are absent from Leishmania, and which are not closely related to the previously described trypanosomal sialidase/trans-sialidase enzyme. We also document the occurrence in Endotrypanum of homologues of the leishmanial surface metalloproteinase gp63 genes. The combined occurrence of sialidase/trans-sialidase activities and gp63 gene homologues in a unique organism has important ramifications for both field and laboratory studies on the biology of trypanosomatids, especially those related to host infection and evolution.

A proteolytic fragment of Trypanosoma cruzi trans-sialidase lacking the carboxyl-terminal domain is active, monomeric, and generates antibodies that inhibit enzymatic activity.

trans-Sialidase isolated from trypomastigote forms of Trypanosoma cruzi, the protozoan parasite that causes Chagas' disease, is multimeric and heterogeneous in size. We show here that limited proteolysis of tans-sialidase with papain yields a single monomeric polypeptide chain of 70 kDa that conserves full enzymatic activity on soluble and membrane-bound substrates. The papain fragment lacks most of the 12-amino acid repeats of the carboxyl-terminal domain that comprises about 50% of the native trans-sialidase. When injected into rabbits, the papain-generated fragment induces antibodies that inhibit trans-sialidase activity and trypomastigote sialylation. The repeats are also not required for the stability of the enzyme or for the correct folding during the biosynthesis in Escherichia coli, but seem essential for trans-sialidase oligomerization. We conclude that trans-sialidase is composed of two structurally and functionally independent domains.

Trypanosoma rangeli sialidase lacks trans-sialidase activity.

Extracts and tissue culture supernatants of axenic forms of T. rangeli were assayed for the presence of sialidase and trans-sialidase activities. Using sialyl(alpha 2-3)lactose, sialyl(alpha 2-6)lactose, poly(alpha 2-8)N-acetylneuraminic acid, fetuin and 4-methylumbelliferyl-N-acetylneuraminic acid as sialic acid donors, and lactose as a sialic acid acceptor, no trans-sialidase activity was detected. Nevertheless, T. rangeli lysates and culture supernatants contain a sialidase that hydrolyzes sialyl(alpha 2-3)lactose, and much less efficiently sialyl(alpha 2-6)lactose, but not poly(alpha 2-8)N-acetylneuraminic acid. T. cruzi trans-sialidase hydrolyzed only sialyl(alpha 2-3)lactose under the same conditions. The T. rangeli and the T. cruzi enzymes differ antigenically and in their pH optimum for hydrolase activity.

Characterization of a novel trans-sialidase of Trypanosoma brucei procyclic trypomastigotes and identification of procyclin as the main sialic acid acceptor.

Here we report the presence of a trans-sialidase on the surface of Trypanosoma brucei culture-derived procyclic trypomastigotes. The enzyme is not detected in lysates of bloodstream trypomastigotes enriched for either stumpy or slender forms. The trans-sialidase catalyzes the transfer of alpha(2-3)-linked sialic acid residues to lactose. beta-galactopyranosyl residues are at least 100 times better acceptors for sialic acid than alpha-galactopyranosyl residues. In the absence of efficient acceptors, the purified enzyme transfers sialic acid to water, i.e., it acts as a sialidase. Although the T. cruzi and T. brucei trans-sialidases have very similar donor and acceptor specificities, they are antigenically distinct. Sodium dodecyl sulfate-polyacramide gel electrophoresis under nonreducing conditions and silver staining of the purified trans-sialidase reveals a single band of 63 kD. When the surface membrane of live procyclic trypomastigotes is trans-sialylated, using radioactive sialyllactose as the donor substrate, it appears that the only sialylated surface molecule is procyclin. Pronase treatment of live parasites removes only part of the surface sialic acid, in agreement with recent data showing that the glycosylphosphatidylinositol anchor of procyclin is sialylated (Ferguson, M. A. J., M. Murray, H. Rutherford, and M. J. McConville. 1993. Biochem. J. In press).

Production of monoclonal antibodies for the identification of a strain of Trypanosoma cruzi.

Mouse monoclonal antibodies (MAb) were produced against antigen from epimastigote forms of the Montalvania 17 strain of Trypanosoma cruzi. Several T. cruzi-specific MAb were obtained, some of which were capable of discriminating between different T. cruzi strains.

Isolation and immunological analysis of Trypanosoma cruzi glycolipids.

A water-soluble glycolipidic fraction from Trypanosoma cruzi was isolated using a mixture of hexane and isopropanol. Analysis by SDS-polyacrylamide gel electrophoresis, after staining by silver and Sudan Black B, showed that the fraction contained one band with a relatively high mobility. Its reactivity and specificity with human chagasic sera and T. cruzi infected mouse sera or with sera from patients with several other pathologies was determined by an immunoradiometric assay. The glycolipid-based radioimmunoassay for the detection of T. cruzi antigens provided a sensitive measure of its activity. However, cross-reactivity with several sera from patients with visceral and cutaneous leishmaniasis was detected.